One of the most important interactions in T cell recognition involves the interplay between the T cell receptor (TCR) and the CD8 coreceptor with a peptide-containing major histocompatibility complex (MHC) class I molecule. This interaction is important for both positive and negative selection in the thymus during T cell differentiation and for activation of mature T cells within the periphery. CD8 is encoded by two distinct but closely linked genes alpha and beta which give rise to either an alpha/alpha homodimer or an alpha/beta heterodimer on the cell surface or to a secreted molecule. While the TCR binds to the polymorphic alpha1 and alpha2 domains of MHC class I, CD8alpha/alpha binds to the nonpolymorphic alpha3 domain. Recently, the crystal structure of the CD8alpha/alpha homodimer was solved showing that CD8 has a structure similar to immunoglobulin. However, it is unclear whether CD8 binds class I MHC in a manner analogous to immunoglobulin; that is, with the loops corresponding to the complementarity determining regions (CDR). Our published work on mutations in CD8alpha indicate that the CDR1 and CDR2 loops are important for binding. In this proposal we plan to perform a more detailed analysis of these loops based on the crystal structure and test other regions of the molecule such as the CDR3 and D-E loops. Another aim is to determine whether the CD8alpha/beta heterodimer is capable of binding to MHC class I and if so, examine the difference in affinity of interaction between each form and MHC class I. To analyze the regions of CD8alpha important for interaction with MHC class I, we plan to perform a mutational analysis of amino acids that are solvent accessible and that are located either in loops or in the framework. In addition, we will test the hypothesis that the amino acids in the CDR1 and CDR2 loops determine species specificity of binding by replacing the murine loops with the human loops. The effect of the mutations on the ability of CD8alpha/alpha to bind to either HLA class I or the non-classical MHC molecule HLA-G will be tested using a transient cell-cell adhesion assay. This assay will also be used to determine if CD8alpha/beta can bind to MHC class I by cotransfecting the cDNAs for the alpha chain and a chimeric beta-alpha chain (extracellular portion is beta) into the COS7 cells and asking whether an anti-CD8beta antibody can block. One of the problems in studying CD8, is that whenever the alpha and beta genes are expressed in the same cell, both alpha/alpha and alpha/beta dimers will be expressed on the cell surface (beta cannot be transported to the cell surface without pairing with alpha). To avoid the difficulties in interpretation, we will create purified soluble forms of these molecules. We can then perform a Scatchard analysis to determine affinity of binding to either MHC class I with beta2-microglobulin or to a peptide corresponding to the negatively charged loop in the alpha3 domain. T cells play an essential role in detecting and destroying virus-infected and malignant cells. The work proposed will extend our understanding of the basic mechanisms of immune recognition, a crucial part of our defense against cancer and infection.